This work was supported by Connaught Fund New Investigator Award to S.P., Canada Foundation for Innovation, NSERC Discovery Grant Program (grants RGPIN-2015-05114 and RGPIN-2020-05881), University of Manchester and University of Toronto Joint Research Fund, and University of Toronto XSeed Program. C.T. was supported by NSERC USRA fellowship.

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The authors disclose no conflict of interests.

The results above demonstrate that the described IR laser assisted micropatterning (microphotopatterning) protocol provides reproducible adherent patterns of various shapes that enables the manipulation of cell shape and cytoskeletal architecture. Although numerous micropatterning methods have been developed both prior to and after the debut of microphotopatterning, this method possesses several advantages. First, it does not require specialized equipment and cleanrooms that are usually only found within Engineering depa...

Cell shape is a key determinant of fundamental biological processes such as tissue morphogenesis1, cell migration2, cell proliferation3, and gene expression4. Changes in cell shape are driven by an intricate balance between dynamic rearrangements of the cytoskeleton that deforms the plasma membrane and extrinsic factors such as external forces exerted on the cell and the geometry of cell-cell and cell-matrix adhesions5. Migrating mesenchymal cells, for instance, polymerize a dense actin network at the leading edge that pushes the plasma membrane forward and creates a wide lamellipodia6, while actomyosin contractility retracts the cell&#39;s narrow trailing edge to detach the cell from its current position7,8. Disrupting signaling events that give rise to such specialized cytoskeletal structures perturbs shape and polarity and slows cell migration9. In addition, epithelial sheet bending during gastrulation requires actomyosin-based apical constriction that causes cells and their neighbors to become wedge-shaped10. Although these studies highlight the importance of cell shape, the inherent heterogeneity in cell shape has encumbered efforts to identify mechanisms that connect morphology to function.
To this end, numerous approaches to manipulate cell shape have been developed over the past three decades. These approaches achieve their goal by either constraining the cell with a three-dimensional mold or controlling cellular adhesion geometry through patterned deposition of extracellular matrix (ECM) proteins onto an antifouling surface, a technique termed micropatterning11. Here we will review a number of techniques that have gained popularity throughout the years.
Originally pioneered as an approach for microelectronic applications, soft lithography-based microcontact printing has unequivocally become a cult favorite12. A master wafer is first fabricated by selectively exposing areas of a photoresist-coated silicon substrate to photoirradiation, leaving behind a patterned surface13. An elastomer, such as PDMS, is then poured onto the master wafer to generate a soft &#34;stamp&#34; that transfers ECM proteins to a desired substrate11,14. Once fabricated, a master wafer can be used to cast many PDMS stamps that give rise to highly reproducible micropatterns12. However, the patterns cannot be readily adjusted due to the lengthy photolithography process. This process also requires highly specialized&#160;equipment and cleanrooms that are not typically available in Biology departments.
More recently, direct printing using deep UV&#160;has been reported to circumvent limitations posed by traditional lithography-based approaches. Deep UV light is directed through a photomask to selective areas of a glass coverslip coated with poly-L-lysine-grafted-polyethylene glycol. Chemical groups exposed to deep UV are photoconverted without the use of photosensitive linkers to enable binding of ECM proteins15. The lack of photosensitive linkers enables patterned coverslips to remain stable at room temperature for over seven months15. This method avoids the use of cleanrooms and photolithography equipment and requires less specialized training. However, the requirement for photomasks still poses a substantial hurdle for experiments that require readily available changes in patterns.
In addition to methods that manipulate cell geometry through controlled deposition of ECM proteins on a 2D surface, other seek to control cell shape by confining cells in 3D microstructures. Many studies have adapted the soft lithography-based approach described above to generate 3D, rather than 2D, PDMS chambers to investigate shape-dependent biological processes in embryos, bacteria, yeast and plants16,17,18,19. Two-photon polymerization (2PP) has also taken the lead as a microfabrication technique that can create complex 3D hydrogel scaffolds with nanometer resolution20. 2PP relies on the principles of two-photon adsorption, where two photons delivered in femtosecond&#160;pulses are absorbed simultaneously by a molecule - photoinitiator in this case - that enables local polymerization of photopolymers21. This technique has been heavily employed to print 3D scaffolds that mimic the native ECM structures of human tissue and has been shown to induce low photochemical damage to cells22.
The debut of microphotopatterning 10 years ago gave researchers the opportunity to fabricate micropatterns while avoiding inaccessible and specialized equipment. Microphotopatterning creates patterns on the micron scale by thermally removing selective regions of poly-vinyl alcohol (PVA) coated on activated glass surfaces using an infrared&#160;laser23,24. ECM proteins that attach only the underlying glass surface and not PVA then serve as biochemical cues to enable controlled spreading dynamics and cell shape. This method also offers superior flexibility since patterns can be readily changed in real time. Here, we provide a step-by-step protocol of microphotopatterning by using a commercial multi-photon imaging system. The described protocol is designed for rapid and automated fabrication of large patterns. We demonstrated that these patterns efficiently control cell shape by constraining the geometry of cell-ECM adhesions. Finally, we demonstrate that the described patterning technique modulates the organization and dynamics of the actin cytoskeleton.

<table><tbody><tr><td>(3-Aminopropyl)trimethoxysilane</td><td>Aldrich</td><td>281778</td><td></td></tr><tr><td>10 cm Cell Culture Dish</td><td>VWR</td><td>10062-880</td><td>Polysterene, TC treated, vented</td></tr><tr><td>25X Apo LWD Water Dipping Objective</td><td>Nikon</td><td>MRD77225</td><td></td></tr><tr><td>3.5 cm Cell Culture Dish</td><td>VWR</td><td>10861-586</td><td>Polysterene, TC treated, vented</td></tr><tr><td>4&#039;,6-Diamidino-2-Phenylindole (DAPI)</td><td>Thermo</td><td>62248</td><td>1mg/mL dihydrochloride solution</td></tr><tr><td>Bovine Serine Albumin</td><td>BioShop</td><td>ALB005</td><td></td></tr><tr><td>Dulbecco&#039;s Phosphate-Buffered Saline</td><td>Wisent</td><td>311-425-CL</td><td></td></tr><tr><td>Ethanolamine</td><td>Sigma-Aldrich</td><td>E9508</td><td></td></tr><tr><td>Fibronectin</td><td>Sigma-Aldrich</td><td>FC010</td><td>1mg/mL in pH 7.5 buffer</td></tr><tr><td>Fibronectin Antibody</td><td>BD</td><td>610077</td><td>Mouse</td></tr><tr><td>Fiji</td><td>ImageJ</td><td></td><td>Version 1.53c</td></tr><tr><td>Fluorescent Phalloidin</td><td>Invitrogen</td><td>A12380</td><td>568nm</td></tr><tr><td>Glass Coverslip</td><td>VWR</td><td>16004-302</td><td>22 &amp;times; 22 mm</td></tr><tr><td>Glutaraldehyde</td><td>Electron Microscopy Sciences</td><td>16220</td><td>25% aqueous solution</td></tr><tr><td>Hydrochloric Acid</td><td>Caledon</td><td>6025-1-29</td><td>37% aqueous solution</td></tr><tr><td>IR Laser</td><td>Coherent</td><td>Chameleon Vision</td><td></td></tr><tr><td>Minimal Essential Medium &amp;alpha;</td><td>Gibco</td><td>12561-056</td><td></td></tr><tr><td>Mounting Medium</td><td>Sigma</td><td>F4680</td><td></td></tr><tr><td>Mouse Secondary Antibody</td><td>Cell Signaling Technology</td><td>4408S</td><td>Goat, 488nm</td></tr><tr><td>Multi-Photon Microscope</td><td>Nikon</td><td>A1R MP+</td><td></td></tr><tr><td>Myosin Light Chain Antibody</td><td>Cell Signaling Technology</td><td>3672S</td><td>Rabbit</td></tr><tr><td>NIS Elements</td><td>Nikon</td><td></td><td>Version 5.21.03</td></tr><tr><td>Nitric Acid</td><td>Caledon</td><td>7525-1-29</td><td>70% aqueous solution</td></tr><tr><td>Photoshop</td><td>Adobe</td><td></td><td>Version 21.2.1</td></tr><tr><td>Pluronic F-127</td><td>Sigma</td><td>P2443</td><td>Powder</td></tr><tr><td>Poly(vinyl alchohol)</td><td>Aldrich</td><td>341584</td><td>MW 89000-98000, 98% hydrolyzed</td></tr><tr><td>Rabbit Secondary Antibody</td><td>Cell Signaling Technology</td><td>4412S</td><td>Goat, 488nm</td></tr><tr><td>Shaker</td><td>VWR</td><td>10127-876</td><td>Alsoknown as analog rocker</td></tr><tr><td>Sodium Borohydride</td><td>Aldrich</td><td>452882</td><td>Powder</td></tr><tr><td>Sodium Hydroxide</td><td>Sigma-Aldrich</td><td>S8045</td><td></td></tr><tr><td>Sodium Phosphate Dibasic</td><td>Sigma</td><td>S5136</td><td>Powder</td></tr><tr><td>Sodium Phosphate Monobasic</td><td>Sigma</td><td>S5011</td><td>Powder</td></tr><tr><td>Spyder</td><td>Anaconda</td><td>4.1.4</td><td></td></tr><tr><td>Trypsin</td><td>Wisent</td><td>325-042-CL</td><td>0.05% aqueous solution with 0.53mM EDTA</td></tr></tbody></table>

control of cell geometry through infrared laser assisted micropatterning

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular compartments while circumventing complications arising from natural cell-to-cell variations. To standardize cell shape, cells are either confined in 3D molds or controlled for adhesive geometry through adhesive islands. However, traditional micropatterning techniques based on photolithography and deep UV etching heavily depend on clean rooms or specialized equipment. Here we present an infrared laser assisted micropatterning technique (microphotopatterning) modified from Doyle et al. that can be conveniently set up with commercially available imaging systems. In this protocol, we use a Nikon A1R MP+ imaging system to generate micropatterns with micron precision through an infrared (IR) laser that ablates preset regions on poly-vinyl alcohol coated coverslips. We employ a custom script to enable automated pattern fabrication with high efficiency and accuracy in systems not equipped with a hardware autofocus. We show that this IR laser assisted micropatterning (microphotopatterning) protocol results in defined patterns to which cells attach exclusively and take on the desired shape. Furthermore, data from a large number of cells can be averaged due to the standardization of cell shape. Patterns generated with this protocol, combined with high resolution imaging and quantitative analysis, can be used for relatively high throughput screens to identify molecular players mediating the link between form and function.

The quality of the experimental data obtained through&#160;micropatterning is largely dependent on the quality of the patterns. To determine the quality of patterns generated with the method above, we first used reflectance microscopy to assess the shape and size of the photo ablated areas of the coverslip. We found that each individual pattern looked very similar to the ablation mask and displayed clear boarders and a surface that reflected light uniformly (Figure 2B). A variety of shapes a...

Watch this Scientific Journal Video about Control of Cell Geometry through Infrared Laser Assisted Micropatterning at JoVE.com

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

The protocol presented here enables automated fabrication of micropatterns that standardizes cell shape to study cytoskeletal structures within mammalian cells. This user-friendly technique can be set up with commercially available imaging systems and does not require specialized equipment inaccessible to standard cell biology laboratories.

Micropatterning is an established technique in the cell biology community used to study connections between the morphology and function of cellular ...

control-cell-geometry-through-infrared-laser-assisted

Research

JoVE Journal

Bioengineering

3.4K Views.  University of Toronto. Micropatterning is een krachtige techniek die door wetenschappers wordt gebruikt om verbanden tussen celvorm en de activiteit van intercellulaire machines te begrijpen. Hoewel verschillende technieken onderzoekers in staat stellen om de celvorm te moduleren, vereisen deze technieken vaak gespecialiseerde apparatuur die niet toegankelijk is voor sommige biologielaboratoria. Deze video begeleidt u door de stappen die nodig zijn om micropatterning te automatiseren op een in de handel verkrijgbaa...

Video: Control of Cell Geometry through Infrared Laser Assisted Micropatterning

Stencil Micropatterning of Human Pluripotent Stem Cells for Probing Spatial Organization of Differentiation Fates

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into all three germ layers. This makes them an attractive cell source for regenerative medicine and experimental modeling of normal and diseased organogenesis. However, the differentiation of hPSCs in vitro is heterogeneous and spatially disordered. Cell micropatterning technologies potentially offer the means to spatially control stem cell microenvironments and organize the resultant differentiation fates. Micropatterning hPSCs needs to take into account the stringent requirements for hPSC survival and maintenance. Here, we describe stencil micropatterning as a method that is highly compatible with hPSCs. hPSC micropatterns are specified by the geometries of the cell stencil through-holes, which physically confine the locations where hPSCs can access and attach to the underlying extracellular matrix-coated substrate. Due to this mode of operation, there is greater flexibility to use substrates that can adequately support hPSCs as compared to other cell micropatterning methods. We also highlight critical steps for the successful generation of hPSC micropatterns. As an example, we demonstrate that stencil micropatterning of hPSCs can be used to modulate spatial polarization of cell-cell and cell-matrix adhesions, which in turn determines mesoendoderm differentiation patterns. This simple and robust method to micropattern hPSCs widens the prospects of establishing experimental models to investigate tissue organization and patterning during early embryonic development.

Human pluripotent stem cells (hPSCs) have the intrinsic ability to differentiate and self-organize into distinct tissue patterns; although this requires the presentation of spatial environmental gradients. We present stencil micropatterning as a simple and robust method to generate biochemical and mechanical gradients for controlling hPSC differentiation patterns.

Human pluripotent stem cells (hPSCs), including embryonic stem cells and induced pluripotent stem cells, have the intrinsic ability to differentiate into ...

Developmental Biology

Free-form Light Actuators &#8212; Fabrication and Control of Actuation in Microscopic Scale

Liquid crystalline elastomers (LCEs) are smart materials capable of reversible shape-change in response to external stimuli, and have attracted researchers&#39; attention in many fields. Most of the studies focused on macroscopic LCE structures (films, fibers) and their miniaturization is still in its infancy. Recently developed lithography techniques, e.g., mask exposure and replica molding, only allow for creating 2D structures on LCE thin films. Direct laser writing (DLW) opens access to truly 3D fabrication in the microscopic scale. However, controlling the actuation topology and dynamics at the same length scale remains a challenge.
In this paper we report on a method to control the liquid crystal (LC) molecular alignment in the LCE microstructures of arbitrary three-dimensional shape. This was made possible by a combination of direct laser writing for both the LCE structures as well as for micrograting patterns inducing local LC alignment. Several types of grating patterns were used to introduce different LC alignments, which can be subsequently patterned into the LCE structures. This protocol allows one to obtain LCE microstructures with engineered alignments able to perform multiple opto-mechanical actuation, thus being capable of multiple functionalities. Applications can be foreseen in the fields of tunable photonics, micro-robotics, lab-on-chip technology and others.

Here, we fabricate 3D polymeric micro/nano structures in which both the shape and the molecular alignment can be engineered with nanometer scale accuracy by the use of direct laser writing. Light induced deformation of several types of liquid crystalline elastomer microstructures can be controlled in the microscopic scale.

Liquid crystalline elastomers (LCEs) are smart materials capable of reversible shape-change in response to external stimuli, and have attracted ...

Engineering

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain central aspects of cellular physiology. To easily combine cell patterning with Multi-Electrode Arrays (MEAs) and silicon-based &#8216;lab on a chip&#8217; technologies, a microfabrication-compatible protocol is required. We describe a method that utilizes deposition of the polymer parylene-C on SiO2&#160;wafers. Photolithography enables accurate and reliable patterning of parylene-C at micron-level resolution. Subsequent activation by immersion in fetal bovine serum (or another specific activation solution) results in a substrate in which cultured cells adhere to, or are repulsed by, parylene or SiO2 regions respectively. This technique has allowed patterning of a broad range of cell types (including primary murine hippocampal cells, HEK 293 cell line, human neuron-like teratocarcinoma cell line, primary murine cerebellar granule cells, and primary human glioma-derived stem-like cells). Interestingly, however, the platform is not universal; reflecting the importance of cell-specific adhesion molecules. This cell patterning process is cost effective, reliable, and importantly can be incorporated into standard microfabrication (chip manufacturing) protocols, paving the way for integration of microelectronic technology.

Cell Patterning on Photolithographically Defined 
Parylene-C: SiO2 Substrates

This protocol describes a microfabrication-compatible method for cell patterning on SiO2. A predefined parylene-C design is photolithographically printed on SiO2 wafers. Following incubation with serum (or other activation solution) cells adhere specifically to (and grow according to the conformity of) underlying parylene-C, whilst being repulsed by SiO2 regions.

Cell patterning platforms support broad research goals, such as construction of predefined in vitro neuronal networks and the exploration of certain ...

Stretching Micropatterned Cells on a PDMS Membrane

Mechanical forces exerted on cells and/or tissues play a major role in numerous processes. We have developed a device to stretch cells plated on a PolyDiMethylSiloxane (PDMS) membrane, compatible with imaging. This technique is reproducible and versatile. The PDMS membrane can be micropatterned in order to confine cells or tissues to a specific geometry. The first step is to print micropatterns onto the PDMS membrane with a deep UV technique. The PDMS membrane is then mounted on a mechanical stretcher. A chamber is bound on top of the membrane with biocompatible grease to allow gliding during the stretch. The cells are seeded and allowed to spread for several hours on the micropatterns. The sample can be stretched and unstretched multiple times with the use of a micrometric screw. It takes less than a minute to apply the stretch to its full extent (around 30%). The technique presented here does not include a motorized device, which is necessary for applying repeated stretch cycles quickly and/or computer controlled stretching, but this can be implemented. Stretching of cells or tissue can be of interest for questions related to cell forces, cell response to mechanical stress or tissue morphogenesis. This video presentation will show how to avoid typical problems that might arise when doing this type of seemingly simple experiment.

This manuscript presents a technique to apply or release forces on adherent cells or tissues using unidirectional stretching.

Mechanical forces exerted on cells and/or tissues  play a major role in numerous processes. We have developed a device to stretch  cells plated on a ...

Micropatterning Transmission Electron Microscopy Grids to Direct Cell Positioning within Whole-Cell Cryo-Electron Tomography Workflows

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules present in the cellular context and preserved in a near-native frozen-hydrated state. However, there are challenges associated with culturing and/or adhering cells onto TEM grids in a manner that is suitable for tomography while retaining the cells in their physiological state. Here, a detailed step-by-step protocol is presented on the use of micropatterning to direct and promote eukaryotic cell growth on TEM grids. During micropatterning, cell growth is directed by depositing extra-cellular matrix (ECM) proteins within specified patterns and positions on the foil of the TEM grid while the other areas remain coated with an anti-fouling layer. Flexibility in the choice of surface coating and pattern design makes micropatterning broadly applicable for a wide range of cell types. Micropatterning is useful for studies of structures within individual cells as well as more complex experimental systems such as host-pathogen interactions or differentiated multi-cellular communities. Micropatterning may also be integrated into many downstream whole-cell cryo-ET workflows, including correlative light and electron microscopy (cryo-CLEM) and focused-ion beam milling (cryo-FIB).

The goal of this protocol is to direct cell adhesion and growth to targeted areas of grids for cryo-electron microscopy. This is achieved by applying an anti-fouling layer that is ablated in user-specified patterns followed by deposition of extra-cellular matrix proteins in the patterned areas prior to cell seeding.

Whole-cell cryo-electron tomography (cryo-ET) is a powerful technology that is used to produce nanometer-level resolution structures of macromolecules ...

Biochemistry

Simple Lithography-Free Single Cell Micropatterning using Laser-Cut Stencils

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single cell resolution. Current state-of-the-art single cell micropatterning techniques involve soft lithography and micro-contact printing, which is a powerful technology, but requires trained engineering skills and certain facility support in microfabrication. These limitations require a more accessible technique. Here, we describe a simple alternative lithography-free method: stencil-based single cell patterning. We provide step-by-step procedures including stencil design, polyacrylamide hydrogel fabrication, stencil-based protein incorporation, and cell plating and culture. This simple method can be used to pattern an array of as many as 2,000 cells. We demonstrate the patterning of cardiomyocytes derived from single human induced pluripotent stem cells (hiPSC) with distinct cell shapes, from a 1:1 square to a 7:1 adult cardiomyocyte-like rectangle. This stencil-based single cell patterning is lithography-free, technically robust, convenient, inexpensive, and most importantly accessible to those with a limited bioengineering background.

This protocol introduces a lithography-free micropatterning method that is simple and accessible to those with a limited bioengineering background. This method utilizes customized laser-cut stencils to micropattern extracellular matrix proteins in a shape of interest for modulating cell morphologies. The procedure for micropatterning is demonstrated using induced pluripotent stem cell derived cardiomyocytes.

Micropatterning techniques have been widely used in cell biology to study effects of controlling cell shape and size on cell fate determination at single ...

A Versatile Method of Patterning Proteins and Cells

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning techniques work well only with simple shapes, small areas and selected bio-materials. This article describes a method to distribute cell suspensions as well as substrate solutions into complex, long, closed (dead-end) polydimethylsiloxane (PDMS) microchannels using negative pressure. This method enables researchers to pattern multiple substrates including fibronectin, collagen, antibodies (Sal-1), poly-D-lysine (PDL), and laminin. Patterning of substrates allows one to indirectly pattern a variety of cells. We have tested C2C12 myoblasts, the PC12 neuronal cell line, embryonic rat cortical neurons, and amphibian retinal neurons. In addition, we demonstrate that this technique can directly pattern fibroblasts in microfluidic channels via brief application of a low vacuum on cell suspensions. The low vacuum does not significantly decrease cell viability as shown by cell viability assays. Modifications are discussed for application of the method to different cell and substrate types. This technique allows researchers to pattern cells and proteins in specific patterns without the need for exotic materials or equipment and can be done in any laboratory with a vacuum.

This report describes a simple, easy to perform technique, using low pressure vacuum, to fill microfluidic channels with cells and substrates for biological research.

Substrate and cell patterning techniques are widely used in cell biology to study cell-to-cell and cell-to-substrate interactions. Conventional patterning ...

Initial 3D Cell Cluster Control in a Hybrid Gel Cube Device for Repeatable Pattern Formations

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its restrictions. Producing few repeatable results of pattern formation deteriorates the analysis of the mechanisms underlying the self-organization. Reducing variation in initial culture conditions, such as the cell density and distribution in the extracellular matrix (ECM), is crucial to enhance the repeatability of a 3D culture. In this article, we demonstrate a simple but robust procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain highly repeatable pattern formations. A micromold with a desired shape was fabricated by using photolithography or a machining process, and it formed a 3D pocket in the ECM contained in a hybrid gel cube (HGC). Highly concentrated cells were then injected in the pocket so that the cell cluster shape matched with the fabricated mold shape. The employed HGC allowed multi-directional scanning by its rotation, which enabled high-resolution imaging and the capture of the entire tissue structure even though a low-magnification lens was used. Normal human bronchial epithelial cells were used to demonstrate the methodology.&#160;

We present a procedure for controlling the initial cell cluster shape in a 3D extracellular matrix to obtain a repeatable pattern formation. A cubic device containing two different hydrogels is employed to achieve multi-directional imaging for tissue pattern formation.

The importance of in vitro 3D cultures is considerably emphasized in cell/tissue culture. However, the lack of experimental repeatability is one of its ...

Control of Cell Adhesion using Hydrogel Patterning Techniques for Applications in Traction Force Microscopy

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat soft substrates that deform under cell traction (2D-TFM). TFM relies on the use of linear elastic materials, such as polydimethylsiloxane (PDMS) or polyacrylamide (PA). For 2D-TFM on PA, the difficulty in achieving high throughput results mainly from the large variability of cell shapes and tractions, calling for standardization. We present a protocol to rapidly and efficiently fabricate micropatterned PA hydrogels for 2D-TFM studies. The micropatterns are first created by maskless photolithography using near-UV light where extracellular matrix proteins bind only to the micropatterned regions, while the rest of the surface remains non-adhesive for cells. The micropatterning of extracellular matrix proteins is due to the presence of active aldehyde groups, resulting in adhesive regions of different shapes to accommodate either single cells or groups of cells. For TFM measurements, we use PA hydrogels of different elasticity by varying the amounts of acrylamide and bis-acrylamide and tracking the displacement of embedded fluorescent beads to reconstruct cell traction fields with regularized Fourier Transform Traction Cytometry (FTTC).
To further achieve precise recording of cell forces, we describe the use of a controlled dose of patterned light to release cell tractions in defined regions for single cells or groups of cells. We call this method local UV illumination traction force microscopy (LUVI-TFM). With enzymatic treatment, all cells are detached from the sample simultaneously, whereas with LUVI-TFM traction forces of cells in different regions of the sample can be recorded in sequence. We demonstrate the applicability of this protocol (i) to study cell traction forces as a function of controlled adhesion to the substrate, and (ii) to achieve a greater number of experimental observations from the same sample.

Near-UV lithography and traction force microscopy are combined to measure cellular forces on micropatterned hydrogels. The targeted light-induced release of single cells enables a high number of measurements on the same sample.

Traction force microscopy (TFM) is the main method used in mechanobiology to measure cell forces. Commonly this is being used for cells adhering to flat ...

Pattern Generation for Micropattern Traction Microscopy

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer length scale allows the use of these patterned contact zones for the measurement of traction forces, as each micropatterned dot allows for the formation of a single focal adhesion that then deforms the soft, underlying hydrogel. This approach has been used for a wide range of cell types, including endothelial cells, smooth muscle cells, fibroblasts, platelets, and epithelial cells. 
This review describes the evolution of techniques that allow the printing of extracellular matrix proteins onto polyacrylamide hydrogels in a regular array of dots of prespecified size and spacing. As micrometer-scale patterns are difficult to directly print onto soft substrates, patterns are first generated on rigid glass coverslips that are then used to transfer the pattern to the hydrogel during gelation. First, the original microcontact printing approach to generate arrays of small dots on the coverslip is described. A second step that removes most of the pattern to leave islands of small dots is required to control the shapes of cells and cell clusters on such arrays of patterned dots. 
Next, an evolution of this approach that allows for the generation of islands of dots using a single subtractive patterning step is described. This approach is greatly simplified for the user but has the disadvantage of a decreased lifetime for the master mold needed to make the patterns. Finally, the computational approaches that have been developed for the analysis of images of displaced dots and subsequent cell-generated traction fields are described, and updated versions of these analysis packages are provided.

We describe improvements to a standard method for measuring cellular traction forces, based on microcontact printing with a single subtractive patterning step of dot arrays of extracellular matrix proteins on soft hydrogels. This method allows for simpler and more consistent fabrication of island patterns, essential for controlling cell cluster shape.

Micropattern traction microscopy allows control of the shape of single cells and cell clusters. Furthermore, the ability to pattern at the micrometer ...

Control of Cell Geometry through Infrared Laser Assisted Micropatterning

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